JP6099045B2 - Triazolopyrimidine derivative compounds - Google Patents

Description

  The present invention relates to a triazolopyrimidine derivative compound.

Fatty acid binding protein 4 (Fatty acid
binding protein-4, FABP4) is an intracellular protein involved in lipid metabolism and insulin sensitivity, and in recent years, a mechanism for promoting inflammatory activity in macrophages has been clarified. FABP4 has attracted attention as a new marker that links the two events of qualitative accumulation and inflammatory response observed in arteriosclerotic lesions, and [3- [2- (3,4-diphenyl-5-ethyl-1H- It has also been reported that FABP4-selective inhibitors including pyrazol-1-yl) phenyl] phenoxyacetic acid (BMS309403) suppress atherosclerosis formation. Therefore, if FABP4 expression can be imaged non-invasively, it is expected to be effective for elucidating the pathology of atherosclerosis and evaluating instability.

Non-Patent Document 1 plans development of a new nuclear medicine molecular imaging probe targeting FABP4, selects a triazolopyrimidine derivative reported to have high affinity for FABP4 as the mother nucleus of the probe, It is described that [ ¹²³ I] TAP1 in which is introduced was designed and synthesized. In Non-Patent Document 1, [ ¹²⁵ I] TAP1 has a dissociation coefficient of 54.2 nM for FABP4, and exhibits higher binding and selectivity to FABP4 than FABP3 and 5, [ ¹²⁵ I] The logP value of TAP1 is 2.7, the radioactivity accumulation of [ ¹²⁵ I] TAP1 in THP-1 cells is inhibited by BMS309403 in a concentration-dependent manner, and [ ¹²⁵ I] TAP1 is specific to intracellular FABP4. As a result of experiments on the distribution of radioactivity in the body, the accumulation of radioactivity in the stomach and thyroid was low, indicating stability against in vivo deiodination, and during arteriosclerosis imaging. Therefore, [ ¹²³ I] TAP1 is used as a FABP4 imaging probe because of its low radioactivity accumulation in the normal aorta, which is the background. It is said to have basic properties.

  Radioactive TAP1 is also described in Patent Document 1.

Japanese Patent Application No. 2013-086848

Journal of the Molecular Imaging Society of Japan, vol. 5, No. 2.103, Publisher: Japan Society for Molecular Imaging, Publication Date: May 24, 2012

However, when the present inventors administered [ ¹²³ I] TAP1 to a WHHLMI rabbit, which is an animal model of arteriosclerosis, [ ¹²³ I] TAP1 is specific for radioactivity accumulation in a lesion (aorta) in vivo. It has become clear that there is still room for improvement. This is presumably because [ ¹²³ I] TAP1 is highly fat-soluble and thus nonspecifically accumulates in normal tissues, the background increases, and the washout becomes slow.

The present invention has been made in view of the above circumstances, and an object thereof is to provide a FABP4 probe that is less lipophilic than [ ¹²³ I] TAP1 and can specifically bind to FABP4.

  According to the present invention, a compound represented by the following general formula (1) or a salt thereof is provided.

In the above formula (1), X is a radioactive fluorine atom, and n is 1 .

  Moreover, according to this invention, the pharmaceutical composition containing the compound of the said Formula (1) description is provided.

  Moreover, according to this invention, the compound represented by following General formula (2) or its salt is provided.

In the above formula (2), R ₁ is a halogen atom, an alkylsulfonic acid ester group, a halogenated alkylsulfonic acid ester group, or an aromatic sulfonic acid ester group which may have a substituent, and n is 1 .

  Further, according to the present invention, the compound represented by the above formula (2) or a salt thereof is reacted with a radioactive fluoride ion, and the above formula (1) (wherein X is a radioactive fluorine atom). A method for producing a radioactive fluorine-labeled organic compound is provided, which comprises a step of synthesizing a compound represented by.) Or a salt thereof.

  According to the present invention, it is possible to provide a FABP4 probe that has low fat solubility and can specifically bind to FABP4.

It is a figure which shows the synthetic scheme of the unlabeled body of the compound which concerns on this invention. FIG. 5 shows 8-anilino-1-naphthalene-sulfonic acid (1,8-ANS) inhibition of FABP4 by a compound according to the present invention. It is a figure which shows the binding inhibitory activity with respect to [< ¹²⁵ > I] TAP1 of the compound based on this invention. It is a figure which shows the synthetic scheme of the labeling precursor of the compound based on this invention. It is a figure which shows the synthetic scheme of the radioactive fluorine labeled body of the compound which concerns on this invention. It is a figure which shows the evaluation result of FABP4 binding selectivity. It is a figure which shows the normal mouse body distribution of [< ¹⁸ > F] FTAP-PEG1. It is a figure which shows the biodistribution of the mouse tumor model of [< ¹⁸ > F] FTAP-PEG1. ^[18 F] is a diagram showing a matching of the distribution of the ^[18 F] FTAP-PEG1 and FABP4 in mouse tumor models of FTAP-PEG1. (A), (c) are autoradiographs of sliced frozen sections of the excised tumor, (b), (d) are FABP4 of adjacent frozen sections (sections adjacent to sections (a), (c)) Immunostaining. It is the figure which evaluated the in-vivo stability of [< ¹⁸ > F] FTAP-PEG1. (A) shows the analysis result of [ ¹⁸ F] FTAP-PEG1 before administration, and (b) shows the analysis result of [ ¹⁸ F] FTAP-PEG1 in the tumor. The result of the western blotting of the C6 cell of a culture state is shown. The result of the western blotting of the tumor extracted from the C6 tumor cell transplant mouse is shown.

(Triazolopyrimidine derivative compound)
Specific examples of the compound according to the present invention include the following compounds.
5-((3- (2-fluoroethoxy) phenoxy) methyl) -2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one [the above general formula ( 1) A compound in which X is a fluorine atom and n is an integer of 1. Hereinafter, it may be abbreviated as “FTAP-PEG1”. ].
5-((3- (2- (2-fluoroethoxy) ethoxy) phenoxy) methyl) -2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one [In the above general formula (1), X is a fluorine atom, and n is an integer of 2. Hereinafter, it may be abbreviated as “FTAP-PEG2”. ].
5-((3- (2- (2- (2-fluoroethoxy) ethoxy) ethoxy) phenoxy) methyl) -2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidine-7 (4H) -one [In the above general formula (1), X is a fluorine atom and n is an integer of 3. Hereinafter, it may be abbreviated as “FTAP-PEG3”. ].

  These compounds according to the present invention may form a salt, and such a salt is included in the present invention in a pharmaceutically acceptable salt.

  In the present invention, “salts” include those derived from inorganic or organic acids, or inorganic or organic bases. Specifically, hydrochloride, hydrobromide, hydroiodic acid, sulfate, nitrate, perchlorate, fumarate, maleate, phosphate, glycolate, lactate, salicylate , Succinate, tartrate, acetate, citrate, methanesulfonate, ethanesulfonate, p-toluenesulfonate, aspartate, glutamate, formate, benzoate, malonate, Examples include, but are not limited to, naphthalene-2-sulfonate, trifluoroacetate, benzenesulfonate, amine salt, and ammonium salt.

  As described above, in the general formula (1), n is an integer of 1 or more and 3 or less, preferably n is an integer of 1 or 2, and more preferably, n is an integer of 1.

  The compound according to the present invention can be synthesized, for example, by the synthetic route shown in the following scheme 1.

  First, a compound (m-hydroxyphenol protected body) is prepared in which only the m-position hydroxy group of m-hydroxyphenol is protected with a protecting group (P) such as benzyl. As the protecting group (P) and its introduction method, various ones described in Green's Protective Groups in Organic Synthesis, P17-245 (Wiley-Interscience; 4th edition) can be adopted.

Also, an ethylene glycol derivative having a repeating number (n) of 1 to 3 in which _one terminal hydroxy group is substituted with a fluoro group and the other terminal hydroxy group is substituted with a leaving group (L ₁ ) is prepared. . When synthesizing FTAP-PEG1, an ethylene glycol derivative having a repeating number (n) of 1 is used, and when synthesizing FTAP-PEG2, an ethylene glycol derivative having a repeating number (n) of 2 is used. In the case of synthesizing PEG3, an ethylene glycol derivative having a repeating number (n) of 3 is used. Examples of the leaving group (L ₁ ) include halogen groups such as chloro, bromo and iodo groups; alkyl sulfonate groups such as methanesulfonate groups; halogenated alkyl sulfonate esters such as trifluoromethanesulfonate groups. A group; an aromatic sulfonic acid ester group which may have a substituent such as a benzenesulfonic acid ester group, a 4-methylbenzenesulfonic acid ester group, or a 4-nitrobenzenesulfonic acid ester group can be used.

  Then, the Sn2 reaction between the prepared ethylene glycol derivative and the m-hydroxyphenol protector is carried out in the presence of a base such as potassium carbonate in an aprotic solvent such as dimethylformamide (step 1).

  Next, the hydroxy group is deprotected for the compound obtained in step 1 (step 2). As the deprotection method, various methods described in Green's Protective Groups in Organic Synthesis, P17-245 (Wiley-Interscience; 4th edition) can be employed.

Thereafter, in the presence of a base such as potassium carbonate in an aprotic solvent such as dimethylformamide, the compound obtained in step ₂ and 5-methyl-2 having a leaving group (L ₂ ) at the 5-position methyl group -Sn2 reaction with phenyl- [1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one derivative is carried out (step 3). Examples of the leaving group (L ₂ ) include halogen groups such as chloro group, bromo group and iodo group; alkylsulfonyl groups such as methanesulfonyl group; halogenated alkylsulfonyloxy groups such as trifluoromethanesulfonyl group; benzenesulfonyl group, 4 -An aromatic sulfonyl group which may have a substituent such as a methylbenzenesulfonyl group or a 4-nitrobenzenesulfonyl group can be used. Thereby, the compound which concerns on this invention can be obtained.

(Radiofluorine-labeled triazolopyrimidine derivative compound)
In the above general formula (1), X is a fluorine atom. In a preferred embodiment, X is a radioactive fluorine atom, and more specifically, F-18 is more preferable. By administering a compound labeled with F-18, FABP4 can be detected non-invasively by positron tomography (PET).

More specifically, the compound of the present invention is preferably a radioactive fluorine-labeled compound shown below.
5-((3- (2- [ ¹⁸ F] fluoroethoxy) phenoxy) methyl) -2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one [ In the general formula (1), X is a radioactive fluorine atom ( ¹⁸ F), and n is an integer of 1. Hereinafter, it may be abbreviated as “[ ¹⁸ F] FTAP-PEG1”. ].
5-((3- (2- (2- [ ¹⁸ F] fluoroethoxy) ethoxy) phenoxy) methyl) -2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidine-7 ( 4H) -one [In the above general formula (1), X is a radioactive fluorine atom ( ¹⁸ F), and n is an integer of 2]. Hereinafter, it may be abbreviated as “[ ¹⁸ F] FTAP-PEG2”. ].
5-((3- (2- (2- (2- [ ¹⁸ F] fluoroethoxy) ethoxy) ethoxy) phenoxy) methyl) -2-phenyl- [1,2,4] triazolo [1,5-a Pyrimidin-7 (4H) -one [In the above general formula (1), X is a radioactive fluorine atom ( ¹⁸ F), and n is an integer of 3]. Hereinafter, it may be abbreviated as “[ ¹⁸ F] FTAP-PEG3”. ].

  When a radioactive compound in which X is a radioactive fluorine atom is employed as the compound according to the present invention in the above general formula (1), the compound represented by the above general formula (2) or a salt thereof is used as a labeling precursor. be able to. As the salt, the salts exemplified above can be adopted.

In the above formula (2), R ₁ is a halogen atom such as a chloro group, a bromo group or an iodo group; an alkyl sulfonate group such as a methanesulfonate group; a halogenated alkylsulfone such as a trifluoromethanesulfonate group An acid ester group; or an aromatic sulfonic acid ester group which may have a substituent, such as a benzenesulfonyloxy group, a 4-methylbenzenesulfonic acid ester group, and 4-nitrobenzenesulfonyloxy group. Preferably, it is an alkylsulfonic acid ester group, a halogenated alkylsulfonic acid ester group, or an aromatic sulfonic acid ester group that may have a substituent, and more preferably, it may have a substituent. An aromatic sulfonic acid ester group, and a 4-methylbenzenesulfonic acid ester group is more preferable.

As described above, in the above formula (2), n is an integer of 1 or more and 3 or less. In the above formula (2), n is 1, a labeled precursor of [ ¹⁸ F] FTAP-PEG1, and in the above formula (2), n is 2, a labeled precursor of [ ¹⁸ F] FTAP-PEG2, The compound in which n is 3 in the above formula (2) can be used as a labeling precursor for [ ¹⁸ F] FTAP-PEG3.

  These labeling precursors can be synthesized, for example, according to the synthesis route of Scheme 2 below.

First, a compound (m-hydroxyphenol protected body) in which only the m-position hydroxy group of m-hydroxyphenol is protected with a protecting group (P ₁ ) such as benzyl is prepared. The protecting group (P ₁ ) and the method for introducing it are described in Green's Protective Groups in Organic Synthesis, pp. Various types described in 16-298 (Wiley-Interscience; 4th edition) can be employed.

In addition, an ethylene glycol derivative having a repeating number (n) of 1 to 3 in which one terminal hydroxy group is substituted with a leaving group (L ₃ ) is prepared. When synthesizing FTAP-PEG1, an ethylene glycol derivative having a repeating number (n) of 1 is used, and when synthesizing FTAP-PEG2, an ethylene glycol derivative having a repeating number (n) of 2 is used. In the case of synthesizing PEG3, an ethylene glycol derivative having a repeating number (n) of 3 is used. Examples of the leaving group (L ₁ ) include halogen groups such as chloro group, bromo group and iodo group; alkyl sulfonic acid ester groups such as methanesulfonic acid ester group; halogenated alkyl sulfonic acids such as trifluoromethanesulfonic acid ester group An ester group; an aromatic sulfonic acid ester group that may have a substituent such as a benzenesulfonic acid ester group, a 4-methylbenzenesulfonic acid ester group, or a 4-nitrobenzenesulfonic acid ester group can be used.

  Then, the Sn2 reaction between the prepared ethylene glycol derivative and the m-hydroxyphenol protector is carried out in the presence of a base such as potassium carbonate in an aprotic solvent such as dimethylformamide (step 1).

Next, a protecting group (P ₂ ) is introduced into the terminal hydroxy group of the compound obtained in step 1 (step ₂ ). The protecting group (P ₂ ) and its introduction method are described in Green's Protective Groups in Organic Synthesis, pp. Various types described in 16-298 (Wiley-Interscience; 4th edition) can be employed.

Next, the phenolic hydroxy group (P ₁ ) is deprotected for the compound obtained in step 2 (step 3). As a method of deprotection, Green's Protective Groups in Organic Synthesis, pp. Various types described in 16-298 (Wiley-Interscience; 4th edition) can be employed.

Thereafter, in the presence of a base such as potassium carbonate in an aprotic solvent such as dimethylformamide, the compound obtained in step 3, and 5-methyl-2 having a leaving group (L ₂ ) at the 5-position methyl group A Sn2 reaction with -phenyl- [1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one is carried out (step 4). Examples of the leaving group (L ₂ ) include halogen groups such as chloro group, bromo group and iodo group; alkyl sulfonic acid ester groups such as methanesulfonic acid ester group; halogenated alkyl sulfonic acids such as trifluoromethanesulfonic acid ester group An ester group; an aromatic sulfonic acid ester group that may have a substituent such as a benzenesulfonic acid ester group, a 4-methylbenzenesulfonic acid ester group, or a 4-nitrobenzenesulfonic acid ester group can be used.

Next, the hydroxy group (P ₂ ) is deprotected for the compound obtained in step 4 (step 5). As a method of deprotection, Green's Protective Groups in Organic Synthesis, pp. Various types described in 16-298 (Wiley-Interscience; 4th edition) can be employed.

Then, a leaving group (R ₁ ) for introducing radioactive fluorine by a nucleophilic substitution reaction is introduced into the hydroxy group (P ₂ ). As the leaving group (R ₁ ), various ones exemplified above can be adopted. When halogen is employed as the leaving group (R ₁ ), a compound in which R _{1 in} formula (2) is a halogen atom by acting thionyl chloride or phosphorus tribromide on the hydroxy group deprotected at step 5 Can be synthesized. When a sulfonic acid ester is employed as the leaving group (R ₁ ), a sulfonic acid anhydride or a sulfonic acid halide is allowed to act on the hydroxy group deprotected at step 5 in the presence of a base such as pyridine. Thus, a compound in which R ₁ in formula (2) is a sulfonate group can be synthesized.

(Synthesis of Radiofluorinated Labeled Triazolopyrimidine Derivative Compound According to the Present Invention)
The radioactive fluorine-labeled triazolopyrimidine derivative compound according to the present invention is synthesized by reacting a compound represented by the general formula (2) or a salt thereof, which is a labeling precursor thereof, with a radioactive fluoride ion. Can do. As the radioactive fluoride ion, [ ¹⁸ F] fluoride ion is preferable. [ ¹⁸ F] fluoride ions can be produced by irradiating [ ¹⁸ O] water with protons accelerated by a cyclotron to generate a nuclear reaction of ¹⁸ O (p, n) ¹⁸ F. The produced [ ¹⁸ F] fluoride ions are obtained by adsorbing target water containing [ ¹⁸ F] fluorine ions to an anion exchange resin and desorbing the [ ¹⁸ F] fluorine ions adsorbed on the resin with an aqueous potassium carbonate solution. Is preferably prepared as [ ¹⁸ F] potassium fluoride. In addition, the radioactive fluorine labeling reaction of the compound represented by the general formula (2) or a salt thereof uses [ ¹⁸ F] potassium potassium prepared above, a phase transfer catalyst such as Cryptofix 222 (trade name), and carbonic acid carbonate. It is preferably carried out in the presence of a base such as potassium.

(Method of using triazolopyrimidine derivative compound according to the present invention)
The triazolopyrimidine derivative compound according to the present invention has high selectivity and high affinity for FABP4. Therefore, among the compounds according to the present invention, the radioactive compound labeled with F-18 can be used as an imaging agent for detecting and visualizing unstable plaque (atherogenic plaque) in vivo.

(Pharmaceutical composition)
In the present invention, the “pharmaceutical composition” can be defined as a formulation containing the compound represented by the above general formula (1) or a salt thereof in a form suitable for administration in vivo. This pharmaceutical composition comprises a compound according to the present invention as an active ingredient, and a pharmacologically acceptable carrier, diluent, emulsion, excipient, extender, binder, wetting agent, disintegrant, and surfactant. , Lubricants, dispersants, buffers, preservatives, solubilizers, preservatives, colorants, stabilizers and the like may be included. The pharmaceutical composition of the present invention can be used in an administration method for oral administration or parenteral administration.

In the present invention, the “radiopharmaceutical composition” is defined as the “pharmaceutical composition” defined above containing, as an active ingredient, a compound or a salt thereof in which X is ¹⁸ F in the general formula (1). can do. The dosage form of this radiopharmaceutical composition is preferably one that can be used for parenteral administration, and is intravenous, intraarterial, topical, intraperitoneal or thoracic, subcutaneous, intramuscular, sublingual An injection that can be used for administration, transdermal administration, rectal administration, and the like is more preferable. Such an injection can be prepared by dissolving the radioactive triazolopyrimidine derivative compound according to the present invention in water, physiological saline, Ringer's solution, or the like.

  The concentration of the radioactive triazolopyrimidine derivative compound in the radiopharmaceutical composition of the present invention may be any concentration that can ensure stability against radiolysis.

  The radiopharmaceutical composition of the present invention can be imaged non-invasively (unstable plaque) by administering it to mammals including humans and imaging it using a PET apparatus. Therefore, it is useful for diagnosis and prevention of acute coronary syndrome (ACS).

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples.
For convenience of explanation, the names of the compounds used in the examples and comparative examples are defined as follows.
FTAP-PEG1: 5-((3- (2-fluoroethoxy) phenoxy) methyl) -2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one [above Compound in which X is a fluorine atom and n is an integer of 1 in General Formula (1)].
FTAP-PEG3: 5-((3- (2- (2- (2-fluoroethoxy) ethoxy) ethoxy) phenoxy) methyl) -2-phenyl- [1,2,4] triazolo [1,5-a] Pyrimidin-7 (4H) -one [a compound in which X is a fluorine atom and n is an integer of 3 in the general formula (1)].
TAP1: 5-((3-iodophenoxy) methyl) -2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one

  In the synthesis examples of each compound described in the following examples and comparative examples, each step in the compound synthesis was repeated a plurality of times as necessary, and an amount necessary for use as an intermediate in other synthesis was secured. . In addition, each of the following examples describes preferred examples and is not intended to limit the scope of the present invention.

In this example, analysis and measurement were performed using the following apparatus unless otherwise specified.
NMR apparatus: JNM-ECS400 JEOL Ltd. LC-MS apparatus: LCMS-2010EV Shimadzu Corporation

Example 1 Synthesis of Non-Radioactive FTAP-PEG1 Non-radioactive FTAP-PEG1 was synthesized by the following steps (FIG. 1).

[Step 1-1]
3-Benzyloxyphenol (0.92 g, 4.6 mmol) and potassium carbonate (1.27 g, 9.2 mmol) were added to dimethylformamide (27 mL), and the mixture was stirred for 10 minutes at room temperature, and then in dimethylformamide (5 mL). Of 2-fluoroethyl-4-methylbenzenesulfonate (1 g, 4.6 mmol) was added. After stirring at 105 ° C. overnight, the solvent was distilled off under reduced pressure. The residue was extracted with ethyl acetate, anhydrous sodium sulfate was added for dehydration, and the solvent was evaporated under reduced pressure. The residue was purified by medium pressure preparative chromatography (ethyl acetate: hexane = 1: 8 (volume ratio) was used as an elution solvent), and 1-benzyloxy-3- (2-fluoroethoxy) benzene (Compound 1: 0.9 g, 3.66 mmol, yield 79.6%).
¹ H-NMR of compound 1 (400 MHz, deuterated chloroform): 7.44-7.31 (m, 5H), 7.21-7.17 (t, 8.472 Hz, 1H), 6.63-6. 53 (m, 3H), 5.05 (s, 2H), 4.81-4.67 (td, 4.351, 47.17Hz, 2H), 4.24-4.15 (td, 4.351) 27.478 Hz, 2H).

[Step 1-2]
Compound 1 (0.9 g, 3.7 mmol) was added to methanol (30 mL) and palladium hydroxide / carbon (0.09 g, 0.64 mmol) was added. The mixture was stirred at room temperature for 1 hour under a hydrogen stream. After the reaction, the mixture was filtered through Celite, and the solvent was distilled off under reduced pressure. The residue was purified by medium pressure preparative chromatography (ethyl acetate: hexane = 1: 3 (volume ratio) as an elution solvent) to quantitatively obtain 3- (2-fluoroethoxy) phenol (compound 2). It was.
Subsequently, Compound 2 (0.67 g, 4.3 mmol) was added to dimethylformamide (35 mL), and potassium carbonate (1.2 g, 8.7 mmol) was added thereto to prepare a Compound 2 solution. Meanwhile, 5- (chloromethyl) -2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one (0.76 g, 2.9 mmol) was added to dimethylformamide (10 mL). ), And after stirring, was added to the prepared Compound 2 solution. After stirring at 85 ° C. overnight, the solvent was distilled off under reduced pressure. The residue was extracted with ethyl acetate, anhydrous sodium sulfate was added for dehydration, and the solvent was evaporated under reduced pressure. The residue was purified by medium pressure preparative chromatography (chloroform: methanol = 20: 1 (volume ratio) as an elution solvent), and non-radioactive FTAP-PEG1 (0.12 g, 0.32 mmol, yield based on compound 2) 11.0%).
LC-MS (APCI) of FTAP-PEG1: m / z 381 (pos), 379 (neg).
¹ H-NMR of FTAP-PEG1 (400 MHz, dimethyl sulfoxide-d6): 8.11 (d, 8.243 Hz, 2H), 7.55-7.53 (m, 3H), 7.27-7.22 (T, 8.014 Hz, 1H), 6.69-6.62 (m, 3H), 6.12 (s, 1H), 5.11 (s, 2H), 4.81-4.19 (m 4H).

Comparative Example 1 Synthesis of Non-Radioactive FTAP-PEG3 Non-radioactive FTAP-PEG3 was synthesized by the following steps (FIG. 1).

[Step 2-1]
3-Benzyloxyphenol (0.65 g, 3.3 mmol) and potassium carbonate (0.91 g, 6.6 mmol) were added to dimethylformamide (15 mL), stirred for 10 minutes at room temperature, and then in dimethylformamide (5 mL). Of 2- [2- (2-fluoroethoxy) ethoxy] ethyl-4-methylbenzenesulfonate (1 g, 3.3 mmol) was added. After stirring at 105 ° C. overnight, the solvent was distilled off under reduced pressure. The residue was extracted with ethyl acetate, anhydrous sodium sulfate was added for dehydration, and the solvent was evaporated under reduced pressure. The residue was purified by medium pressure preparative chromatography (ethyl acetate: hexane = 1: 3 (volume ratio) was used as an elution solvent) and purified by 1-benzyloxy-3- (2- (2- (2-fluoroethoxy)). Ethoxy) ethoxybenzene (Compound 3: 0.87 g, 2.6 mmol, yield 78.5%) was obtained.
¹ H-NMR of compound 3 (400 MHz, deuterated chloroform): 7.44-7.32 (m, 5H), 7.19-7.15 (t, 8.014 Hz, 1H), 6.59-6. 51 (m, 3H), 5.04 (s, 2H), 4.63-4.49 (m, 2H), 4.12-4.10 (t, 4.280Hz, 2H), 3.89- 3.84 (t, 5.037 Hz, 2H), 3.80-3.70 (m, 6H).

[Step 2-2]
Compound 3 (0.87 g, 2.6 mmol) was added to methanol (30 mL) and palladium hydroxide / carbon (0.08 g, 0.57 mmol) was added. The mixture was stirred at room temperature for 1 hour under a hydrogen stream. After the reaction, the mixture was filtered through Celite, and the solvent was distilled off under reduced pressure. The residue was purified by medium pressure preparative chromatography (ethyl acetate: hexane = 1: 2 (volume ratio) was used as an elution solvent), and 3- (2- (2- (2-fluoroethoxy) ethoxy) ethoxy) Phenol (compound 4: 0.61 g, 2.5 mmol, yield 96.2%) was obtained.
¹ H-NMR of compound 4 (400 MHz, deuterated chloroform): 7.11-7.07 (t, 8.243 Hz, 1H), 6.48-6.41 (m, 3H), 5.57 (s, aH), 4.62-4.48 (m, 2H), 4.10-4.08 (t, 4.809 Hz, 2H), 3.86-3.83 (t, 4.809 Hz, 2H), 3.80-3.70 (m, 6H).

[Step 2-3]
Compound 4 (0.54 g, 2.2 mmol) was added to dimethylformamide (20 mL), and potassium carbonate (0.62 g, 4.5 mmol) was added thereto to prepare a dimethylformamide solution of compound 4. Meanwhile, 5-chloromethyl-2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one (0.39 g, 1.5 mmol) was added to dimethylformamide (10 mL). In addition, after stirring, it was added to the compound 4 solution prepared above. After stirring at 85 ° C. overnight, the solvent was distilled off under reduced pressure. The residue was extracted with ethyl acetate, anhydrous sodium sulfate was added for dehydration, and the solvent was evaporated under reduced pressure. The residue was purified by medium pressure preparative chromatography (chloroform: methanol = 10: 1 (volume ratio) was used as an elution solvent), and non-radioactive FTAP-PEG3 (0.18 g, 0.38 mmol, yield 25.3). %).
LC-MS (APCI) of FTAP-PEG3: m / z 469 (pos), 467 (neg).
¹ H-NMR of FTAP-PEG3 (400 MHz, dimethyl sulfoxide-d6): 8.14-8.12 (d, 7.327 Hz, 2H), 7.55-7.53 (m, 3H), 7.25 -7.21 (t, 8.014 Hz, 1H), 6.66-6.59 (m, 3H), 6.11 (s, 1H), 5.10 (s, 2H), 4.57-4 .44 (m, 2H), 4.10-4.08 (t, 4.351 Hz, 2H), 3.75-3.58 (m, 8H).

(Example 2 ) 1,8-ANS inhibition experiment on FABP4 The non-radioactive FTAP-PEG1 obtained in Example 1 and the non-radioactive FTAP-PEG3 obtained in Comparative Example 1 are specific for FABP4. In order to confirm whether or not it has binding affinity, a competitive inhibition experiment was conducted using 8-anilino-1-naphthalene-sulfonic acid (1,8-ANS), which is a fluorescent FABP4-binding compound, as a competitive ligand. 10 mmol / L phosphate buffer pH 7.4 (hereinafter also abbreviated as “PB”) (0.12 mL) and FABP4 (Cayman; 0.5 mmol / L (PB solution), 0.075 mL) And 1,8-ANS (Tokyo Chemical Industry Co., Ltd .; 6 mmol / L, 0.2 volume% ethanol-containing PB solution), 0.075 mL), and gradually diluted to 0.25, 0.061,. 015, 0.0038, 0.00095, 0.000228, 0.000057, 0.00001
Each non-radioactive FTAP-PEG1 solution in dimethyl sulfoxide (0.03 mL) adjusted to a concentration of 5 mg / mL was added, and each was incubated at room temperature for 5 minutes. Thereafter, 0.09 mL of each sample was applied to a 96-well plate, and the fluorescence intensity was measured using TECAN Infinite M200 PRO (excitation wavelength: 370 nm, fluorescence wavelength: 475 nm).
For FTAP-PEG3, 0.30, 0.075, 0.019, 0.0047, 0.0012, 0.00028 was substituted for the non-radioactive FTAP-PEG1 solution in dimethyl sulfoxide (0.03 mL). A non-radioactive FTAP-PEG3 solution in dimethyl sulfoxide (0.03 mL) adjusted to concentrations of 0.000072 and 0.0000187 mg / mL was used in the same manner.
The results are shown in FIG. As a result of the examination, the inhibition constant (Ki) calculated from the competitive inhibition experiment with 1,8-ANS was Ki = 68.0 nM for FTAP-PEG1 and Ki = 485 nM for FTAP-PEG3.

Synthesis of (Example ¹⁾ [125 I] Synthesis of TAP1 ^[125 I] TAP1 was synthesized according to the following steps.

[Step 3-1]
Aminoguanidine bicarbonate (11.6 g, 85.4 mmol) was added to pyridine (112 mL) under ice-cooling, and benzoyl chloride (10 g, 71.1 mmol) was added thereto little by little. After stirring the mixed solution at room temperature for 20 hours, the solvent was distilled off under reduced pressure, water (58 mL) and 2 mol / L sodium hydroxide (46 mL) were added to the residue, and the mixture was stirred at room temperature for 24 hours. After completion of the reaction, the obtained precipitate was collected by suction filtration to obtain N-benzamidoguanidine (4.76 g, 26.7 mmol, yield 37.6%).
¹ H-NMR of N-benzamidoguanidine (400 MHz, dimethyl sulfoxide-d6): 10.25 (br, s, 1H), 7.93-7.91 (m, 2H), 7.26-7.25 ( m, 3H), 6.82 (br, s, 2H), 6.69 (br, s, 2H).

[Step 3-2]
N-benzamidoguanidine (4.76 g, 26.7 mmol) obtained in Step 3-1 was added to 40 mL of water and heated to reflux at 110 ° C. for 6 hours. After completion of the reaction, the reaction solution was distilled off under reduced pressure to obtain 3-phenyl-1H-1,2,4-triazol-5-amine (3.0 g, 18.7 mmol, yield 70.1%).
¹ H-NMR (400 MHz, dimethyl sulfoxide-d6) of 3-phenyl-1H-1,2,4-triazol-5-amine: 12.11 (br, s, 1H), 7.88-7.86 ( d, 2H, J = 8.78 Hz), 7.40-7.36 (m, 2H), 7.33-7.29 (m, 1H), 6.05 (br, s, 2H).

[Step 3-3]
3-Phenyl-1H-1,2,4-triazol-5-amine (0.95 g, 5.93 mmol) obtained in Step 3-2 was dissolved in 15 mL of acetic acid, and ethyl 4-chloroacetoacetate (1.07 g) was dissolved. 6.52 mmol) and stirred at 80 ° C. overnight. After completion of the reaction, the precipitate was filtered with suction, and 5-methyl-2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one (1.0 g, 3. 86 mmol; yield 65.1%).
¹ H-NMR of 400-methyl-2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one (400 MHz, dimethyl sulfoxide-d6): 8.12 ( s, 2H), 8.12-8.10 (m, 2H), 7.55-7.53 (m, 3H), 6.20 (s, 1H), 4.68 (s, 2H).

[Step 3-4]
3-Bromophenol (0.34 g, 1.53 mmol) and potassium carbonate (0.21 g, 1.53 mmol) were added to dimethylformamide (7 mL) to prepare a 3-bromophenol solution. 5-Methyl chloride-2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one (0.20 g, 0.767 mmol) obtained in step 3-3 Dissolved in dimethylformamide (3 mL), added to the prepared 3-bromophenol solution, and stirred at 85 ° C. for 19 hours. After the reaction, the solvent was distilled off under reduced pressure, and the residue was extracted with ethyl acetate. After dehydrating by adding anhydrous sodium sulfate to the extracted ethyl acetate, the solvent was distilled off under reduced pressure, and the resulting residue was subjected to medium pressure preparative chromatography (chloroform: methanol = 10: 1 (volume ratio)) as an elution solvent. ) And 5-((3-bromophenoxy) methyl) -2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one (0.12 g, 0 314 mmol, yield 40.9%).
LC-MS (ESI) of 5-((3-bromophenoxy) methyl) -2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one: m / z 397 (pos), 395 (neg).

[Step 3-5]
5-((3-Bromophenoxy) methyl) -2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one (0. 05 g, 0.113 mmol) was dissolved in dimethylformamide (5 mL), triethylamine (2.5 mL) was added, and then bistributyltin (0.13 g, 0.226 mmol) and tetrakis (triphenylphosphine) palladium (0) (0 0.013 g, 0.0113 mmol) was added, and the mixture was heated to reflux at 100 ° C. for 4 hours. After the reaction, the solvent was distilled off under reduced pressure, and chloroform was added to the residue to dissolve it. After purification by medium pressure preparative chromatography (chloroform: methanol = 10: 1 (volume ratio) was used as an elution solvent), a thin layer was further obtained. Purification was performed by chromatography (chloroform: methanol = 10: 1 (volume ratio) was used as a developing solvent), and 2-phenyl-5-((3-tributylstannylphenoxy) methyl)-[1,2,4 ] Triazolo [1,5-a] pyrimidin-7 (4H) -one (0.016 g, 0.026 mmol; yield 23.1%) was obtained.
LC-MS (ESI) of 2-phenyl-5-((3-tributylstannylphenoxy) methyl)-[1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one: m / Z 607 (neg).

[Step 3-6]
2-Phenyl-5-((3-tributylstannylphenoxy) methyl)-[1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one obtained in step 3-5 ( To 1 mg, 0.0016 mmol), 1% by volume acetic acid-containing methanol (0.4 mL), N-chlorosuccinimide (2.4 mg), and methanol (4.8 mL) were added to prepare a TAP1-labeled precursor solution. Then, the prepared TAP1-labeled precursor solution (0.163 mL) was added to a methanol solution of N-chlorosuccinimide (0.5 mg / mL, 0.044 mL), and [ ¹²⁵ I] sodium iodide aqueous solution (37 MBq,. 02 mL) was added and stirred at room temperature for 30 minutes. A saturated aqueous sodium sulfite solution (0.01 mL) was added to the reaction solution to terminate the reaction. After the solvent was distilled off, ethyl acetate (0.5 mL) and water (0.5 mL) were added to carry out a liquid separation operation. . After recovering the ethyl acetate phase, sodium sulfate (2.5 g) was added for dehydration, the solvent was distilled off under reduced pressure, and methanol (0.15 mL) was added to form a methanol solution. The obtained methanol solution was subjected to HPLC (total liquid unit LC20-A manufactured by Shimadzu Corporation, column 5C18-AR-II, mobile phase 0.1% (v / v) trifluoroacetic acid-containing acetonitrile: 0.1% (v / v). Trifluoroacetic acid aqueous solution = 63: 37 (volume ratio), flow rate 1.5 ml / min), and a peak with a retention time of 36.08 minutes was identified as the fraction of [ ¹²⁵ I] TAP1 and fractionated. Ethyl acetate (0.5 mL) was added to the preparative solution to carry out a liquid separation operation. After recovering the ethyl acetate phase, 2.5 g of sodium sulfate was added for dehydration, and the solvent was distilled off under reduced pressure. Methanol (0.2 mL) Was added to prepare a [ ¹²⁵ I] TAP1 solution as a methanol solution.

The labeling yield of [ ¹²⁵ I] TAP1 was 24% radiochemical yield, and the radiochemical purity was 99% or more. The radiochemical purity of [ ¹²⁵ I] TAP1 was analyzed by HPLC under the conditions shown below. Further, it was confirmed that the retention time was the same as that of the non-radioactive TAP1 synthesized by the method described later (step 4).
HPLC analysis conditions:
Column COSMOSIL (registered trademark, Nacalai Tesque, Inc.) (5C18-ARII 10 × 250 mm ID)
Column temperature Room temperature mobile phase 0.1% (v / v) trifluoroacetic acid aqueous solution: 0.1% (v / v) acetonitrile containing trifluoroacid = 37/63
Flow rate 1.5mL / min detection RI DETECTOR US-3000T (Universal Giken)
Injection volume 200μL
Retention time 17.8 minutes

Reference Example 2 Synthesis of non-radioactive TAP1 [Step 4]
3-Iodinated phenol (0.34 g, 1.53 mmol) and potassium carbonate (0.21 g, 1.53 mmol) were added to dimethylformamide (7 mL) to prepare a 3-iodophenol solution. To 3 mL of dimethylformamide was added 5-methyl-2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one (0.20 g, obtained in Step 3-3 above). 0.767 mmol) was added and stirred to dissolve, then added to the prepared 3-iodophenol solution and stirred at 85 ° C. for 19 hours. After the reaction, the solvent was distilled off under reduced pressure, the residue was extracted with ethyl acetate, anhydrous sodium sulfate was added to the extracted ethyl acetate for dehydration, and the solvent was distilled off under reduced pressure. The obtained residue was purified by medium pressure preparative chromatography (chloroform: methanol = 10: 1 (volume ratio) as an elution solvent), and non-radioactive TAP1 (0.11 g, 0.25 mmol; yield 31). 9%).
¹ H-NMR of non-radioactive TAP1 (400 MHz, dimethyl sulfoxide-d6): 8.12-8.10 (m, 2H), 7.54-7.46 (m, 3H), 7.43-7.42 (T, 1H, J = 1.832 Hz), 7.36-7.33 (dt, 1.374, 7.327 Hz, 1H), 7.13-7.06 (m, 2H), 5.96 ( s, 1H), 5.05 (s, 2H).
LC-MS (ESI) of non-radioactive TAP1: m / z 445 (pos), 443 (neg).

(Example 3) ^[125 I] TAP1 binding inhibition activity of the confirmation FABP4 immobilized by a relative, was saturated experiment was synthesized in Reference Example 1 ^[125 I] TAP1. First, two types of buffers used in this example were prepared with the following compositions.
-Protein binding buffer:
50 mmol / L monosodium dihydrogen phosphate aqueous solution 300 mmol / L sodium chloride aqueous solution 10 mmol / L imidazole aqueous solution pH = 8
・ Interaction buffer
50 mmol / L monosodium dihydrogen phosphate aqueous solution 300 mmol / L aqueous sodium chloride solution 10 mmol / L imidazole aqueous solution 0.005 vol% polysorbate (trade name: Tween 80) aqueous solution pH = 8
Further, non-radioactive TAP1 synthesized by the method shown in Reference Example 1 was adjusted to concentrations of 0.045, 0.009, 0.0018, and 0.00036 mg / mL with an interaction buffer.
Further, non-radioactive FTAP-PEG1 synthesized by the method shown in Example 1 was adjusted to concentrations of 0.096, 0.038, 0.0077, 0.0015, and 0.00031 mg / mL with an interaction buffer.
Non-radioactive FTAP-PEG3 synthesized by the method shown in Comparative Example 1 was adjusted to concentrations of 0.118, 0.047, 0.0095, 0.0019, and 0.00038 mg / mL with an interaction buffer.
In a reaction tank (Eppendorf tube), protein-binding buffer (0.5 mL), Ni-NTA beads (manufactured by QIAGEN, Ni-NTA Magnetic Agarose Beads, 20 μL), and FABP4 aqueous solution (Cayman, 0.75 mg / mL, 2 μL), incubated at room temperature for 1 hour, and after removal of the supernatant, 1 wt% BSA (bovine serum albumin) buffer solution (Cayman, 1 wt% BSA-containing protein binding buffer) was added and room temperature was 30 minutes. Incubated at After removing the supernatant, the above prepared non-radioactive TAP1 (0.045, 0.009, 0.0018, 0.00036 mg / mL), non-radioactive FTAP-PEG1 (0.096, 0.038, 0.0077, 0 .0015, 0.00031 mg / mL) or non-radioactive FTAP-PEG3 (0.118, 0.047, 0.0095, 0.0019, 0.00038 mg / ml) and [ ¹²⁵ I] TAP1 ( Each solution (0.05 mL) mixed with 13.2 kBq) and an interaction buffer (0.4 mL) were added, and incubation was performed at room temperature for 1 hour. In the non-specific binding group, non-radioactive TAP1 (0.09 mg / mL, 10 vol% dimethyl sulfoxide-containing interaction buffer, 0.05 mL) was added simultaneously. After removing the supernatant, washing was performed with 5% by volume ethanol buffer (5% by volume ethanol-containing protein binding buffer), and then the radioactivity of the reaction vessel was measured using an autowell gamma counter (Auto Well Gamma System, ARC-380CL, ARC-2000, manufactured by ALOKA) was used.

The results are shown in Table 1 and FIG. The 50% inhibition concentration (IC ₅₀ ) calculated from the competitive inhibition experiment with [ ¹²⁵ I] TAP1 was 0.3 μmol / L for TAP1, 0.45 μmol / L for FTAP-PEG1, and 5.50 for FTAP-PEG3. It was 0 μmol / L.

(Example 4 ) Confirmation of cell membrane permeability of FTAP-PEG1 and FTAP-PEG3 Whether FTAP-PEG1 and FTAP-PEG3 were accessible to intracellular FABP4 was confirmed by evaluating cell membrane permeability. The cell membrane permeability was evaluated using a BD Gentest ™ precoated PAMPA plate system (BD Biosciences).
First, non-radioactive FTAP-PEG1 synthesized by the method shown in Example 1 was added to 0.038, 0.076 mg / 2.5% phosphate buffered saline (PBS (−) buffer) containing 2.5% by volume of dimethyl sulfoxide. Prepared to a concentration of mL.
Further, non-radioactive FTAP-PEG3 synthesized by the method shown in Comparative Example 1 was adjusted to a concentration of 0.047 and 0.094 mg / mL with a PBS (−) buffer solution containing 2.5% by volume of dimethyl sulfoxide.
The non-radioactive FTAP-PEG1 (0.038, 0.076 mg / mL) and non-radioactive FTAP-PEG3 (0.047, 0...) Prepared above were added to the receiver plate (donor plate) of the BD Genest ™ precoated PAMPA plate system. 094 mg / mL) solution (0.2 mL) was injected. Phosphate buffered saline (2.5 mL) containing 2.5% by volume dimethyl sulfoxide was injected into a filter plate (acceptor plate), and both plates were set, followed by incubation at room temperature for 4 hours. Thereafter, in addition to the original solution, the absorbance (280 nm) of both plates was measured, and the cell membrane permeability (Pe) was determined from the cell membrane permeability calculation formula shown in the following formula (1).

As a result, FTAP-PEG1 was Pe = 1.5 × 10 ⁻⁶ cm / s, and FTAP-PEG3 was Pe = 0.9 × 10 ⁻⁶ cm / s.

(Example 5) Synthesis radioactive ^FTAP-PEG1 radioactive FTAP-PEG1 labeled precursors ([18 F] FTAP-PEG1 ) labeling precursor was synthesized by the following steps (Fig. 4).

[Step 5-1]
3-Benzyloxyphenol (2.0 g, 10 mmol) and potassium carbonate (4.0 g, 30 mmol) were added to dimethylformamide (60 mL), stirred for 10 minutes at room temperature, and then 2-bromo in dimethylformamide (10 mL). Ethanol (3.8 g, 30 mmol) was added. After stirring at 105 ° C. overnight, the solvent was distilled off under reduced pressure. The residue was extracted with ethyl acetate, anhydrous sodium sulfate was added for dehydration, and the solvent was evaporated under reduced pressure. The residue was purified by medium pressure preparative chromatography (ethyl acetate: hexane = 1: 8 (volume ratio) was used as an elution solvent), and 2- (3-benzyloxy) phenoxyethanol (Compound 5, 1.8 g, 7. 4 mmol, yield 74.0%).
¹ H-NMR (400 MHz, deuterated chloroform) of Compound 5: 7.44-7.33 (m, 5H), 7.21-7.17 (t, 8.472 Hz, 1H), 6.62-5. 05 (m, 3H), 4.07-4.05 (m, 2H), 3.97-3.93 (m, 2H).

[Step 5-2]
Compound 5 (1.8 g, 7.4 mmol) was added to dichloromethane (20 mL), and tert-butyldimethylchlorosilane (1.8 g, 11.8 mmol) and imidazole (1.0 g, 14.8 mmol) were added thereto at room temperature. Stir overnight. After evaporating the solvent under reduced pressure, chloroform was added to the residue, and the residue was purified by medium pressure preparative chromatography (ethyl acetate: hexane = 1: 8 (volume ratio) was used as an elution solvent) and purified by 2- (3-benzyloxy). ) Phenoxyethyl-tert-butyldimethylsilyl ether (Compound 6, 2.0 g, 5.6 mmol, yield 75.7%) was obtained.
¹ H-NMR (400 MHz, deuterated chloroform) of Compound 6: 7.44-7.32 (m, 5H), 7.19-7.14 (t, 8.243 Hz, 1H), 6.59-6. 51 (m, 3H), 5.04 (s, 2H), 4.02-4.00 (t, 4.809 Hz, 2H), 3.97-3.94 (t, 4.580 Hz, 2H), 0.91 (s, 9H), 0.10 (s, 6H).

[Step 5-3]
Compound 6 (2.0 g, 5.6 mmol) was added to methanol (40 mL) and palladium hydroxide / carbon (0.2 g) was added. The mixture was stirred at room temperature for 3 hours under a hydrogen stream. After the reaction, the mixture was filtered through Celite, and the solvent was distilled off under reduced pressure. The residue was purified by medium pressure preparative chromatography (ethyl acetate: hexane = 1: 8 (volume ratio) as an elution solvent) and purified by 3- (2-tert-butyldimethylsiloxyethoxy) phenol (compounds 7, 1 0.4 g, 5.2 mmol, yield 93.2%).
¹ H-NMR (400 MHz, deuterated chloroform) of Compound 7: 7.13-7.09 (t, 8.472 Hz, 1H), 6.50-6.41 (m, 3H), 4.02-3. 94 (m, 4H), 0.91 (s, 9H), 0.10 (s, 6H).

[Step 5-4]
Compound 7 (0.96 g, 3.6 mmol) was added to dimethylformamide (40 mL), and potassium carbonate (0.99 g, 7.2 mmol) was added thereto to prepare a compound 7 solution. Meanwhile, 5- (chloromethyl) -2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one (0.63 g, 2.4 mmol) was added to dimethylformamide (10 mL). ), And after stirring, was added to the compound 7 solution prepared above. After stirring at 85 ° C. overnight, the solvent was distilled off under reduced pressure. The residue was extracted with ethyl acetate, anhydrous sodium sulfate was added for dehydration, and the solvent was evaporated under reduced pressure. The residue was purified by medium pressure preparative chromatography (chloroform: methanol = 10: 1 as an elution solvent), and 5- (3- (2-tert-butyldimethylsiloxyethoxy) phenoxy) methyl-2-phenyl- [1,2,4] Triazolo [1,5-a] pyrimidin-7 (4H) -one (Compound 8, 0.52 g, 1.0 mmol, yield 41.7%) was obtained.
LC-MS (APCI) of compound 8: m / z 493 (pos), 491 (neg).

[Step 5-5]
Compound 8 (0.25 g, 0.51 mmol) was added to tetrahydrofuran (10 mL), and tetra-n-butylammonium fluoride (0.16 g, 0.61 mmol) was added little by little on ice. After stirring overnight at room temperature, the solvent was distilled off under reduced pressure, and the residue was purified by medium pressure preparative chromatography (chloroform: methanol = 10: 1 was used as an elution solvent) and purified by 5- (3- (2-hydroxy). Ethoxy) phenoxy) methyl-2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one (compound 9, 0.22 g, 0.57 mmol, 100% yield) Got.
¹ H-NMR of compound 9 (400 MHz, dimethyl sulfoxide-d6): 8.13-8.11 (dd, 8.243, 1.374 Hz, 2H), 7.54-7.48 (m, 3H), 7.22-7.18 (t, 8.701 Hz, 1H), 6.62-6.55 (m, 3H), 5.96 (s, 1H), 5.02 (s, 2H), 3. 98-3.96 (t, 4.809 Hz, 2H), 3.18-3.14 (m, 2H).

[Step 5-6]
Compound 9 (0.22 g, 0.57 mmol) was added to dichloromethane (10 mL), where p-toluenesulfonyl chloride (0.16 g, 0.86 mmol), triethylamine (0.12 g, 1.14 mmol), N, N -Dimethyl-4-aminopyridine (0.007 g, 0.057 mmol) was added in portions on ice. After stirring at room temperature overnight, the solvent was distilled off under reduced pressure. The residue was extracted with ethyl acetate, anhydrous sodium sulfate was added for dehydration, and the solvent was evaporated under reduced pressure. The residue was purified by medium pressure preparative chromatography (chloroform: methanol = 10: 1 (volume ratio) was used as an elution solvent), and 5- (3- (2- (p-toluenesulfonyloxy) ethoxy) phenoxy) Methyl-2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one (FTAP-PEG1 labeled precursor, 0.13 g, 0.23 mmol, yield 41.2 %).
LC-MS (APCI): m / z 533 (pos), 531 (neg).
¹ H-NMR of FTAP-PEG1 labeled precursor (400 MHz, dimethyl sulfoxide-d6): 8.14-8.11 (d, 10.076, 2H), 7.81-7.79 (d, 8.246 Hz) 2H), 7.55-7.47 (m, 5H), 7.23-7.19 (t, 7.785 Hz, 1H), 6.67-6.49 (m, 3H), 6.11 (S, 1H), 5.09 (s, 2H), 4.34-4.32 (m, 2H), 4.17-4.15 (m, 2H), 2.41 (s, 3H)

( Comparative Example 2 ) Synthesis of radioactive FTAP-PEG3 labeled precursor A labeled precursor of radioactive FTAP-PEG3 ([ ¹⁸ F] FTAP-PEG3) was synthesized by the following steps (FIG. 4).

[Step 6-1]
3-Benzyloxyphenol (2.0 g, 10 mmol) and potassium carbonate (4.0 g, 30 mmol) were added to dimethylformamide (60 mL), stirred for 10 minutes at room temperature, and then 2- [ 2- (2-Chloroethoxy) ethoxy] ethanol (5.1 g, 30 mmol) was added. After stirring at 80 ° C. overnight, the solvent was distilled off under reduced pressure. The residue was extracted with ethyl acetate, anhydrous sodium sulfate was added for dehydration, and the solvent was evaporated under reduced pressure. The residue was purified by medium pressure preparative chromatography (ethyl acetate: hexane = 1: 1 as the eluting solvent) and 2- (2- (2- (3-benzyloxy) phenoxy) ethoxy) ethoxy) ethanol (compound 10, 2.5 g, 7.5 mmol, yield 75.0%).
¹ H-NMR (400 MHz, deuterated chloroform) of Compound 10: 7.43-7.30 (m, 5H), 7.19-7.15 (t, 8.243 Hz, qH), 6.60-6. 52 (m, 3H), 5.04 (s, 2H), 4.13-4.10 (t, 5.266 Hz, 2H), 3.86-3.84 (t, 5.266 Hz, 2H), 3.74-3.70 (m, 6H), 3.62-3.61 (t, 4.58 Hz, 2H).

[Step 6-2]
Compound 10 (2.5 g, 7.5 mmol) was added to dichloromethane (20 mL), and tert-butyldimethylchlorosilane (1.8 g, 11.8 mmol) and imidazole (1.0 g, 14.8 mmol) were added thereto at room temperature. Stir overnight. After evaporating the solvent under reduced pressure, the residue was extracted with ethyl acetate, dehydrated by adding anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure. The residue was purified by medium pressure preparative chromatography (ethyl acetate: hexane = 1: 3 as elution solvent) and 2- (2- (2- (3-benzyloxy) phenoxy) ethoxy) ethoxy) ethyl-tert -Butyldimethylsilyl ether (Compound 11, 3.2 g, 7.2 mmol, yield 96.0%) was obtained.
Subsequently, compound 11 (3.2 g, 7.2 mmol) was added to methanol (50 mL) and palladium hydroxide / carbon (0.3 g) was added. The mixture was stirred at room temperature for 2 hours under a hydrogen stream. After the reaction, the mixture was filtered through Celite, and the solvent was distilled off under reduced pressure. The residue was purified by medium pressure preparative chromatography (ethyl acetate: hexane = 1: 3 (volume ratio) was used as an elution solvent) and purified by 3- (2- (2- (2- (tert-butyldimethylsiloxy)). Ethoxy) ethoxy) ethoxy) phenol (compound 12, 1.3 g, 3.7 mmol, yield 50.7%) was obtained.
¹ H-NMR (400 MHz, deuterated chloroform) of Compound 12: 7.05-7.01 (t, 7.556 Hz, 1H), 6.41-6.34 (m, 3H), 4.04-4. 02 (t, 4.630 Hz, 2H), 3.77-3.75 (t, 4.579 Hz, 2H), 3.72-3.70 (t, 5.266 Hz, 2H), 3.65-3 .49 (m, 6H), 0.82 (s, 9H), -0.00229 (s, 6H).

[Step 6-3]
Compound 12 (1.3 g, 3.6 mmol) was added to dimethylformamide (50 mL), and potassium carbonate (0.99 g, 7.2 mmol) was added thereto to prepare a compound 12 solution. Meanwhile, 5- (chloromethyl) -2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one (0.63 g, 2.4 mmol) was added to dimethylformamide (10 mL). ), And after stirring, was added to the compound 12 solution prepared above. After stirring at 85 ° C. overnight, the solvent was distilled off under reduced pressure. The residue was extracted with ethyl acetate, anhydrous sodium sulfate was added for dehydration, and the solvent was evaporated under reduced pressure. The residue was purified by medium pressure preparative chromatography (chloroform: methanol = 10: 1 as an elution solvent) and (3- (2- (2- (2- (tert-butyldimethylsiloxy) ethoxy) ethoxy) Ethoxy) phenoxy) methyl-2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one (compound 13, 0.51 g, 0.88 mmol, relative to compound 12 Yield 36.5%).
LC-MS (APCI) of compound 12: m / z 581 (pos), 579 (neg).
¹ H-NMR (400 MHz, deuterated chloroform) of Compound 12: 8.28-8.26 (d, 9.159 Hz, 2H), 7.46-7.44 (m, 3H), 7.19-7. 15 (t, 8.014 Hz, 1H), 6.59-6.43 (m, 1H), 6.06 (s, 1H), 5.01 (s, 2H), 4.10-4.08 ( t, 4.580 Hz, 2H), 3.87-3.84 (t, 4.580 Hz, 2H), 3.80-3.77 (t, 5.953 Hz, 2H), 3.73-3.58 (M, 6H), 0.88 (s, 9H), 0.05 (s, 6H).

[Step 6-4]
Compound 13 (0.10 g, 0.17 mmol) was added to tetrahydrofuran (5 mL), and tetra-n-butylammonium fluoride (0.05 g, 0.21 mmol) was added little by little on ice. After stirring at room temperature overnight, the solvent was distilled off under reduced pressure, and the residue was purified by medium pressure preparative chromatography (chloroform: methanol = 10: 1 was used as an elution solvent) and (3- (2- (2- (2- (2-Hydroxyethoxy) ethoxy) ethoxy) phenoxy) methyl-2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one (Compound 14, 0.08 g, 0 .17 mmol, 100% yield).
¹ H-NMR (400 MHz, dimethyl sulfoxide-d6) of Compound 14: 8.13-8.11 (d, 6.869 Hz, 2H), 7.51-7.46 (m, 3H), 7.21- 7.17 (t, 8.701 Hz, 1H), 6.62-6.55 (m, 3H), 5.89 (s, 1H), 4.99 (s, 2H), 4.08-4. 07 (t, 4.58 Hz, 2H), 3.74-3.72 (t, 4.58 Hz, 2H), 3.59-3.43 (m, 8H).

[Step 6-5]
Compound 14 (0.08 g, 0.17 mmol) was added to 10 mL of dichloromethane, and p-toluenesulfonyl chloride (0.03 g, 0.16 mmol), triethylamine (0.03 g, 0.34 mmol), N, N-dimethyl was added thereto. -4-Aminopyridine (0.0002 g, 0.017 mmol) was added in portions on ice. After stirring at room temperature overnight, the solvent was distilled off under reduced pressure. The residue was extracted with ethyl acetate, anhydrous sodium sulfate was added for dehydration, and the solvent was evaporated under reduced pressure. The residue was purified by medium pressure preparative chromatography (chloroform: methanol = 10: 1 as an elution solvent) and (3- (2- (2- (2- (p-toluenesulfonyloxy) ethoxy) ethoxy) Phenoxy) methyl-2-phenyl- [1,2,4] triazolo [1,5-a] pyrimidin-7 (4H) -one (FTAP-PEG3 labeled precursor, 0.02 g, 0.03 mmol, yield 20 0.0%).
LC-MS (APCI) of FTAP-PEG3 labeled precursor: m / z 621 (pos), 619 (neg).
¹ H-NMR (400 MHz, deuterated chloroform) of FTAP-PEG3-labeled precursor: 8.25-8.23 (d, 8.243 Hz, 2H), 7.79-7.78 (d, 6.411 Hz, sH ), 7.46-7.28 (m, 5H), 7.23-7.19 (t, 8.701 Hz, 1H), 6.62-6.53 (m, 3H), 6.06 (s) 1H), 5.06 (s, 2H), 4.18-4.11 (m, 4H), 3.85-3.82 (t, 4.351 Hz, 2H), 3.74-3.66 (M, 6H), 2.41 (s, 3H).

(Example 6 ) Labeled synthesis of radioactive FTAP-PEG1 Labeled synthesis of radioactive FTAP-PEG1 ([ ¹⁸ F] FTAP-PEG1) was performed in the following steps (FIG. 5).
First, 4,7,13,16,21,24-hexaoxa-1,10-diazobicyclo [8.8.8] hexacosane (15 mg) and [ ¹⁸ F] potassium fluoride (2000 MBq, 0.1 mL) aqueous solution, A 66 mmol / L potassium carbonate (0.1 mL) aqueous solution was added, vortexed, acetonitrile (0.3 mL) was added, and heating was repeated 3 times at 120 ° C. under a nitrogen gas stream. Thereafter, a labeled precursor (2 mg) of FTAP-PEG1 synthesized in Example 5 was added to dimethyl sulfoxide (0.25 mL), and the mixture was heated at 130 ° C. for 10 minutes. Thereafter, the solvent was replaced with acetonitrile using Sep-Pak (registered trademark) C18 manufactured by Waters. The obtained acetonitrile solution of [ ¹⁸ F] FTAP-PEG1 was HPLC (apparatus: LC-20A, manufactured by Shimadzu Corporation; column: 5C18-AR-II, 10 × 250 mm; eluent: acetonitrile (0.1% by volume) TFA-containing) / water (0.1 vol% TFA-containing) (volume ratio) = 50/50 (0 min) 70/30 to (40 min) gradient) by preparative purified, the desired ^[18 F] FTAP -A fraction of PEG1 was obtained, water was distilled off, ethyl acetate was added, liquid separation was performed, the organic phase was recovered, passed through sodium sulfate, ethyl acetate was distilled off, and acetonitrile was added. The product was obtained as an acetonitrile solution (174 MBq). In order to confirm the purity of the obtained [ ¹⁸ F] FTAP-PEG1, HPLC analysis was performed under the following conditions.

HPLC analysis conditions:
Column COSMOSIL®, 5C18-ARI, 10 × 250 mm D. Column temperature, room temperature eluent, manufactured by Nacalai Tesque Co., Ltd. A solution: 0.1% (v / v) trifluoromethanesulfonic acid-containing water, B solution: 0.1% (v / v) trifluoromethanesulfonic acid-containing acetonitrile mobile phase Gradient flow rate 1.5 mL / min detection over 40 minutes from A / B: 50/50 (volume ratio) to A / B: 30/70 (volume ratio) RI DETECTOR US-3000T (Universal Giken)
Injection volume 200μL
Retention time 21 minutes

As a result, [ ¹⁸ F] FTAP-PEG1 obtained by the above method had a radiochemical yield of 17.3% and a radiochemical purity of 98% or more. [ ¹⁸ F] FTAP-PEG1 was identified by confirming that the retention time was the same as that of the non-radioactive FTAP-PEG1 synthesized in Example 1 under the above HPLC conditions. The obtained [ ¹⁸ F] FTAP-PEG1 was concentrated to remove acetonitrile, and then dissolved in a physiological saline solution containing 0.1% by volume polyoxyethylene sorbitan monooleate (trade name tween 80). Used in examples.

( Comparative Example 3 ) Labeled synthesis of radioactive FTAP-PEG3 Labeled synthesis of radioactive FTAP-PEG 3 ([ ¹⁸ F] FTAP-PEG 3 ) was performed in the following steps (FIG. 5).
First, 4,7,13,16,21,24-hexaoxa-1,10-diazabicyclo [8.8.8] hexacosane (15 mg) and [ ¹⁸ F] potassium fluoride aqueous solution (966 MBq, 0.1 mL), 66 mmol / L potassium carbonate aqueous solution (0.1 mL) was added, vortexed, acetonitrile (0.3 mL) was added, and heating was repeated 3 times at 120 ° C. under a nitrogen gas stream. Thereafter, a labeled precursor (2 mg) of FTAP-PEG3 synthesized in Comparative Example 2 was added to dimethyl sulfoxide (0.25 mL), and the mixture was heated at 130 ° C. for 10 minutes. Thereafter, the solvent was replaced with acetonitrile using Sep-Pak (registered trademark) C18 manufactured by Waters. The obtained acetonitrile solution of [ ¹⁸ F] FTAP-PEG3 was HPLC (apparatus: LC-20A, manufactured by Shimadzu Corporation; column: 5C18-AR-II, 10 × 250 mm;
Mobile phase: acetonitrile (containing 0.1% by volume TFA) / water (containing 0.1% by volume TFA) (volume ratio) = 50/50 (0 minute) to 70/30 (40 minute) gradient) Purify and obtain the desired [ ¹⁸ F] FTAP-PEG3 fraction. After distilling off the water, ethyl acetate was added to carry out a liquid separation operation. The organic phase was recovered, passed through sodium sulfate, and ethyl acetate. After distilling off, acetonitrile was added to obtain the desired product as an acetonitrile solution (2.7 MBq). In order to confirm the purity of the obtained [ ¹⁸ F] FTAP-PEG3, HPLC analysis was performed.

HPLC analysis conditions:
Column COSMOSIL®, 5C18-ARII 10 × 250 mm D. , Nacalai Tesque Co., Ltd. Column temperature Room temperature eluent A solution: 0.1% (v / v) trifluoromethanesulfonic acid-containing water, B solution: 0.1% (v / v) trifluoromethanesulfonic acid-containing acetonitrile mobile phase A / B: 50/50 (volume ratio) to A / B: 30/70 (volume ratio) over 40 minutes Gradient flow rate 1.5 mL / min detection RI DETECTOR US-3000T (Universal Giken)
Injection volume 200μL
Retention time 21 minutes

As a result, [ ¹⁸ F] FTAP-PEG3 obtained by the above method had a radiochemical yield of 23.5% and a radiochemical purity of 98% or more. [ ¹⁸ F] FTAP-PEG3 was identified by confirming that the retention time was the same as that of the non-radioactive FTAP-PEG1 synthesized in Comparative Example 1 under the above HPLC conditions. The obtained [ ¹⁸ F] FTAP-PEG3 was concentrated to remove acetonitrile and then dissolved in a physiological saline solution containing 0.1% by volume polyoxyethylene sorbitan monooleate (trade name tween 80). Used in examples.

(Example 7) ^[18 F] FTAP-PEG1 and ^[18 F] logP value measurement of ^{FTAP-PEG3 [18 F] FTAP} -PEG1 and ^[18 F] Hydrophobic greatly affects the pharmacokinetics or the like of FTAP-PEG3 In order to evaluate about, logP value was measured.
[ ¹⁸ F] FTAP-PEG1 tween 80-containing physiology obtained by the method shown in Example 6 in a mixed solution of phosphate buffered saline (PBS (−)) (3.0 mL) and octanol (3.0 mL). A saline solution (0.004 MBq, 20 μL) or a [ ¹⁸ F] FTAP-PEG3 physiological saline solution containing tween 80 (0.004 MBq, 20 μL) obtained by the method shown in Comparative Example 3 was added, vortexed, and centrifuged. (1000 g, 5 minutes), and 0.5 mL each was collected in three tubes from each of the octanol phase and the aqueous phase. Furthermore, octanol (2 mL) and PBS (-) (3 mL) were added to the remaining octanol phase (1 mL), vortexed, centrifuged (1000 g, 5 minutes), and again 0.5 mL from each of the octanol phase and the aqueous phase Each was collected in 3 tubes. Each tube was measured with a gamma counter (1480 Automatic Gamma Counter Wizard 3, manufactured by Perkin Elmer), and the log P value was obtained by taking the logarithm of the value obtained by dividing the average of the octanol phase radioactivity by the average of the aqueous phase radioactivity. Asked. In addition, instead of [ ¹⁸ F] FTAP-PEG1 or [ ¹⁸ F] FTAP-PEG3, the same operation was performed using [ ¹²⁵ I] TAP1 synthesized in Reference Example 1, and the Log P value of [ ¹²⁵ I] TAP1 was also determined. Asked.

The results are shown in Table 2. Table 2 also shows the result of cLogP obtained with AcDLabs 6.0 for reference. As shown in Table ^2, [18 F] logP value of FTAP-PEG1 is ^1.66, a 1.14 logP value of [18 F] ^FTAP-PEG3, than logP value 2.74 of [125 I] TAP1 Also, it was shown that the hydrophilicity is high.

The binding selectivity evaluation of (Example 8) ^[18 F] immobilized FABP4 binding selectivity evaluation of FTAP-PEG1 ^[18 F] FABP4 of FTAP-PEG1, compare the binding to the a FABP3,5 FABP4 subtype It was done by doing.
First, a protein binding buffer and an interaction buffer were prepared with the same composition as in Example 3 . Protein reaction buffer (Eppendorf tube), protein binding buffer (0.5 mL), Ni-NTA beads (Ni-NTA Magnetic Agarose Beads, QIAGEN) (20 μL), and FABP4 solution (0.75 mg / mL, 50 mmol / L) Phosphate buffer, pH = 7.2, 20% by volume glycerol, 100 mmol / L sodium chloride, Cayman) and FABP3, 5 solution (0.50, 0.70 mg / mL, 50 mmol / L phosphate, respectively) Buffer, pH = 7.2, 20 volume% glycerol, containing 100 mmol / L sodium chloride, Cayman) 2 μL, incubated at room temperature for 1 hour, and after removing the supernatant, 1 wt% BSA (bovine serum albumin) Buffer (containing 1 wt% BSA (Protein binding buffer) was added and incubated at room temperature for 30 minutes. After removal of the supernatant, [ ¹⁸ F] FTAP-PEG1-containing physiological saline solution (0.081 MBq, 0.1 mL) and interaction buffer (0.4 mL) obtained by the method shown in Example 6 were added, and room temperature was added. Incubation was performed for 2 hours. The non-specific binding group consisted of a 10% by volume dimethyl sulfoxide / interaction buffer solution of non-radioactive FTAP-PEG1 synthesized by the method shown in Example 1 (0.0043 mg / mL), 10% by volume dimethyl sulfoxide / interaction buffer (0. 05 mL) was added simultaneously. After removing the supernatant, washing was performed with 5% by volume ethanol buffer (5% ethanol-containing protein binding buffer), and then the radioactivity in the reaction vessel was measured using an autowell gamma counter (Auto Well Gamma System, ARC-380CL, ARC-2000, manufactured by Aroka Co., Ltd.).

The results are shown in FIG. As a result, it showed a higher affinity to FABP4 compared to ^FABP3,5, showed FABP4 selectivity of [18 F] FTAP-PEG1.

Example 9 Normal Mouse Biodistribution Experiment The in vivo radioactivity distribution in normal mice was determined by the organ excision method. Normal mice (ddy, male 5 weeks of age) were administered with the saline solution of [ ¹⁸ F] FTAP-PEG1 in tween 80 obtained by the method shown in Example 6 (0.074 MBq, 0.1 mL). At each time point (n = 3-4) of 5, 15, 30, 60, and 120 minutes, the mice were sacrificed under anesthesia, and organs were removed and organ weights and organ radioactivity levels were measured. Radioactivity measurement was performed using a gamma counter (1480 Automatic Gamma Counter Wizard 3, manufactured by Perkin Elmer). The radioactivity distribution rate of each organ relative to the dose of radioactivity was calculated and determined, and the in vivo radioactivity distribution of [ ¹⁸ F] FTAP-PEG1 was determined.

The results are shown in FIG. The vertical axis | shaft of FIG. 7 shows% dose only for the stomach, and others show% dose / g. As shown in FIG. 7, [ ¹⁸ F] FTAP-PEG1 had a low radioactivity accumulation in bone, indicating stability against in vivo defluorination reaction. In addition, radioactivity accumulation in the normal aorta, which becomes the background during arteriosclerosis imaging, was also low.

(Example 10 ) Confirmation of in vivo FABP4 binding of [ ¹⁸ F] FTAP-PEG1 using mouse tumor model Using mice transplanted with rat glioma cell C6 (obtained from Human Science Research Resource Bank), [ ¹⁸ F] Confirmation of whether FTAP-PEG1 has FABP4 binding ability even in vivo was determined by the same organ excision method as in Example 9 . Normal mice (ddy, male 5 weeks old) were injected with rat glioma cells C6 (obtained from Human Science Research Resource Bank) subcutaneously in the right lower limb region of 2.0 × 10 ⁶ mice, and subjected to the experiment two weeks later. Obtained by the method shown in Example 6 ^[18 F] tween80 containing saline solution FTAP-PEG1 (0.12MBq, 0.1mL) each time 5,30,60,120,180 minutes of tail vein administration of At point (n = 3-4), the animals were sacrificed under anesthesia, and organs were removed and organ weights and organ radioactivity levels were measured. Radioactivity measurement was performed using a gamma counter (1480 Automatic Gamma Counter Wizard 3, manufactured by Perkin Elmer). The radioactivity distribution rate of each organ relative to the dose of radioactivity was calculated and determined, and the in vivo radioactivity distribution of [ ¹⁸ F] FTAP-PEG1 was determined.

The results are shown in FIG. The vertical axis | shaft of FIG. 8 shows% dose only for the stomach, and others show% dose / g. As a result of in vivo radioactivity distribution experiments, the amount of [ ¹⁸ F] FTAP-PEG1 accumulated in C6 tumors infiltrated with macrophages expressing FABP4 increased with time. Moreover, the accumulation ratio with respect to muscles and blood tended to increase, indicating that it has binding ability to FABP4 even in vivo.

Example 11 Confirmation of Consistency of Distribution between [ ¹⁸ F] FTAP-PEG1 and FABP4 Using the C6 tumor cell-transplanted mouse prepared in Example 10 , the intratumoral distribution of [ ¹⁸ F] FTAP-PEG1 was FABP4. In order to confirm that the distribution was reflected, the immunostaining of FABP4 and autoradiography (ARG) of [ ¹⁸ F] FTAP-PEG1 were compared. [ ¹⁸ F] FTAP-PEG1-containing tween 80-containing saline solution (92.5 MBq, 0.1 mL) obtained by the method shown in Example 6 was administered from the tail vein of C6 tumor cell-transplanted mice. 180 minutes after administration of [ ¹⁸ F] FTAP-PEG1, the mice were sacrificed under anesthesia, and the tumors were removed and rapidly frozen in liquid nitrogen (or dry ice / acetone). The frozen tumor tissue was sliced at a thickness of 10 μm to prepare a frozen section. Serial sections were used for ARG and immunostaining. ARG is an imaging plate (BAS-MS) manufactured by FUJIFILM Corporation.
) Was exposed to light for 2.5 hours, and then imaged and analyzed with an image analysis apparatus (BAS 2500) manufactured by FUJIFILM Corporation. For immunostaining, polyclonal antibody AF3150 against anti-human FABP4 (manufactured by R & D Systems) was used as the primary antibody, and simple stain MAX PRO (G) (manufactured by Nichirei) was used as the secondary antibody.

The results are shown in FIG. FIGS. 9A and 9C are ARGs of frozen sections of the excised tumor, and FIGS. 9B and 9D are FABP4 immunostaining of adjacent frozen sections. In FIG. 9 (a), a and b are parts with high radioactivity accumulation, and the same part is also a good part in FIG. 9 (b). In FIG. 9 (c), c and d are sites with high radioactivity accumulation, and the same site is also a well-affected site in FIG. 9 (d). [ ¹⁸ F] FTAP-PEG1 is shown to be consistent in the localization of ARG and FABP4 immunostaining, and [ ¹⁸ F] FTAP-PEG1 is a compound capable of reflecting the distribution of FABP4. confirmed.

(Example 12 ) Confirmation of in vivo accumulation specificity of [ ¹⁸ F] FTAP-PEG1 Using the C6 tumor cell-transplanted mouse prepared in Example 10 , the presence of [ ^The presence or absence of influence of blood radioactivity of ¹⁸ F] FTAP-PEG1 was confirmed. [ ¹⁸ F] FTAP-PEG1-containing tween 80-containing saline solution (3.7 MBq, 0.1 mL) obtained by the method shown in Example 6 was administered from the tail vein of C6 tumor cell-transplanted mice. 180 minutes after administration, death from decapitated blood loss under anesthesia, or transcardial perfusion with physiological saline for 3 minutes, and then each organ including tumor tissue was removed, and the organ weight and radioactivity were determined using a gamma counter. It was measured.

  The results are shown in Table 4. As a result, there is no significant change in the amount of tumor radioactivity accumulated, and the contribution of blood radioactivity to tumor accumulation is considered to be small.

(Example 13) ^[18 F] with C6 tumor cells transplanted mice prepared in confirmation Example 10 in vivo stability of the FTAP-PEG1, that tumor accumulation reflects FABP4 ^{binding, [18} F] It was confirmed by evaluating the in vivo stability in the tumor tissue where FTAP-PEG1 accumulates. [ ¹⁸ F] FTAP-PEG1 in tween 80-containing saline solution (185 MBq, 0.1 mL) obtained by the method shown in Example 6 was administered from the tail vein of C6 tumor cell-transplanted mice. 180 minutes after administration, the mice were sacrificed under anesthesia, and the tumor was removed. : PBS (-) (phosphate buffered saline) = 20: 1: 79, and then homogenate and sonication under ice-cooling. After vortexing with methanol (3 mL), ultracentrifugation (10,000 × g, 5 minutes) was performed, and the supernatant was filtered with a membrane filter (0.45 μm), followed by HPLC under the analysis conditions described in Example 6. Analysis was performed and compared with the analysis results of [ ¹⁸ F] FTAP-PEG1 before administration.

The results are shown in FIG. FIG. 10 (a) shows the analysis result of [ ¹⁸ F] FTAP-PEG1 before mouse administration, and FIG. 10 (b) shows the analysis result of [ ¹⁸ F] FTAP-PEG1 in the tumor. As shown in the figure, the HPLC chart of radioactivity in the tumor is almost equivalent to that of [ ¹⁸ F] FTAP-PEG1 before administration, and no new peak is detected, so [ ¹⁸ F] FTAP- PEG1 was stably present in vivo, at least in tumors, suggesting that it is in a state of binding to FABP4.

(Example 14 ) Confirmation of expression of FABP4 By performing FABP4 Western blotting of tumors excised from C6 cells used in Example 10 in a cultured state or C6 tumor cell-transplanted mice prepared in Example 10 , Expression was confirmed. As a positive control, RT4 cells which are FABP4 multi-expressing cells were used.
Sample preparation for Western blotting of cultured cells was performed as follows. Rat glioma cells in culture on dish C6 (available from Humanity emissions Sa sciences Research Resources Bank), and, after recovering the human bladder carcinoma derived cell cultures RT4 (obtained from American Type Culture Collection) with a scraper, PBS (-) ( 10 mL), centrifuged (1000 g, 5 minutes), and after removing the supernatant, Passive lysis buffer (Promega) (× 5): Protease inhibitor (Product name: Protease inhibitor Cocktail with use mammian and tissue Extracts (manufactured by Sigma): PBS (−) (phosphate buffered saline) = 20: 1: 79 (volume ratio) mixed solution (hereinafter solution A) (1 mL) was added. After ultrasonic crushing, it was centrifuged at 13200 rpm for 30 minutes, and the supernatant was recovered. After protein quantification, solution A was added to adjust to 2.5 and 1.25 mg / mL. To this solution was added 5 × sample buffer (reduced) (0.25 mmol / L Tris-HCl (pH 6.8) 25 mL, SDS 2 g, sucrose 5 g,
Purified water (25 mL) was added to dilute to 2, 1 mg / mL.
The sample for Western blotting of the removed transplanted tumor was prepared as follows. The transplanted tumor excised by the method of Example 10 , solution A (0.5 mL) was added, and after ultrasonic disruption, centrifuged at 13200 rpm for 30 minutes, and the supernatant was collected. After protein quantification, solution A was added to adjust to 1.25 mg / mL. 5 × sample buffer (reduction) was added to this solution to dilute to 1 mg / mL. Two samples of tumors were used. The fat extracted together with the tumor by the method of Example 10 as a positive control and the muscle extracted together with the tumor by the method of Example 10 as a negative control were similarly adjusted.
In Western blotting, the sample prepared above was heated at 100 ° C. for 5 minutes, and then electrophoresed using a PAGEL gel (E-T520L, Ato) (90 minutes, 400 V, 40 mA). After the electrophoresis, a gel was set on the transfer and transferred to a PVDF membrane (Millipore, Immobilon Transfer Membranes) (45 minutes, 20 V, 500 mA). After the transfer, the PVDF membrane was washed with PBS (−) (hereinafter TPBS) containing 0.05% by volume Tween 20 (polyoxyethylene sorbitan monolaurate) for 30 minutes, and then Block One (manufactured by Nacalai Tesque) was added for 30 minutes at room temperature. Blocking was carried out by incubating with. To this PVDF membrane, 1 mL of a solution obtained by diluting an anti-mouse FABP4 rabbit monoclonal antibody (D25B3, Cell Signaling) 1000-fold with TPBS containing 5% by volume of BSA was added as a primary antibody and incubated overnight in a refrigerator. On the other hand, as the primary antibody for β-actin, anti-β-actin mouse monoclonal antibody (A5441, SIGMA) was similarly diluted 1000 times. After washing with TPBS, HRP-labeled anti-rabbit IgG (# 7074, Cell Signaling) or HRP-labeled anti-mouse IgG1 (STAR81P, Serotec) was used as the secondary antibody. After incubation at room temperature for 45 minutes, it was washed with TPBS. Chemi-Lumi One Super (manufactured by Nacalai Tesque) was used as a coloring reagent, and imaging was performed with a gel imaging device (ChemDoc, Bio-Rad).

  The results are shown in FIGS. FIG. 11 shows the results of Western blotting of C6 cells in culture. FIG. 12 shows the results of Western blotting of tumors excised from C6 tumor cell-transplanted mice. In FIG. 12, a and b are tumors, c is muscle, and d is fat. As a result, it was revealed that C6 cells in culture were not expressed in FABP4 but expressed in transplanted tumors. Since macrophages express FABP4, it is considered that FABP4 expressed in the transplanted tumor is derived from macrophages infiltrating into the tumor.

  The compound according to the present invention can be used in the technical field of diagnostic agents, particularly diagnostic imaging agents.

Claims (5)

A compound represented by the following general formula (1) or a salt thereof.
[Wherein, X is a radioactive fluorine atom, and n is 1 . ] The pharmaceutical composition containing the compound represented by following General formula (1).
[Wherein, X is a radioactive fluorine atom, and n is 1 . ] The pharmaceutical composition according to claim 2 , which is used for imaging vulnerable plaque. A compound represented by the following general formula (2) or a salt thereof.
Wherein, R ₁ is a halogen atom, an alkyl sulfonic acid ester group, a halogenated alkylsulfanyl Ho phosphate ester group, or an optionally substituted aromatic sulfonic acid ester radical, n is 1 It is. ] A method for producing a radioactive fluorine-labeled organic compound comprising the step of reacting the compound or a salt thereof according to claim 4 with a radioactive fluoride ion to synthesize a compound represented by the following formula (1) or a salt thereof: .
[Wherein, X is a radioactive fluorine atom, and n is 1 . ]